The past two or three decades have established a seminal role for Protein Tyrosine Phosphatases (PTPs) in human health and disease (Alonso et al., 2004; Andersen et al., 2004). Gene mutations in members of this family result in metabolic, neurological, muscle wasting, and autoimmune diseases, and over 30 have been implicated in the etiology of various cancers (Alonso et al., 2004).
PTPs are a family of approximately 100 phosphatases characterized by their highly conserved CX5R (SEQ ID NO:2) catalytic motif. Once thought to dephosphorylate only phosphotyrosine residues, a subset of PTP's are now known to dephosphorylate phosphoserine- and phosphothreonine-containing proteins, as well as to use RNA, and phosphoinositides as substrates in vitro and in vivo (Tonks et. al., 2001; Li et al., 2000; Andersen et al., 2001; Fauman et al., 1996; Jackson et al., 2001). A structural study of the tumor suppressor phosphatase PTEN revealed a wider active site cleft and suggested positions of key basic amino acids in the P-loop (CKAGKGR) (SEQ ID NO:1) among the reasons for its ability to use the phosphoinositide PI(3,4,5)P3 as its preferred substrate (Lee et al., 1999). Consistent with this observation, other PTP's possessing highly similar or identical active site motifs, including the PTEN homologs PTEN 2 and TPIP, bacterial effector phosphatases SopB and IpgD, and inositol polyphosphate 4 phosphatases 1 and 2, have also now been shown to possess activity against phosphoinositide substrates (Morris et al., 2000; Walker et al., 2001; Wu et al., 2001; Niebuhr et al., 2002; Norris et al., 1998; Bansal et al., 1990; Norris et al., 1994).
Phosphatidylinositol (PtdIns), an abundant membrane phospholipid, is capable of being phosphorylated on the 3, 4, and 5 positions of its inositol ring to form seven unique lipid signaling molecules collectively termed phosphoinositides (PI's) (Vanhaesebroeck et al., 2001). PI's regulate critical cellular functions, including apoptosis, membrane trafficking, cytoskeletal rearrangement, metabolism, growth, and differentiation, by altering the subcellular location, state of aggregation, and activity of a variety of cellular enzymes. PI regulation by lipid kinases, phosphatases, and lipases is therefore critical in achieving proper cellular responses to outside stress (Vanhaesebroeck et al., 2001; Wishart et al., 2002). PI(5)P (phosphatidylinositol-5 phosphate) is the least characterized PI, having only recently been identified as an endogenous lipid (Rameh et al., 1997). Recent studies report changes in intracellular PI(5)P levels during cell cycle progression, as well as upon thrombin treatment and osmotic stress (Morris et al., 2000; Clarke et al., 2001; Meijer et al., 2001). Furthermore, the PHD (plant homeodomain)-containing ING2 protein, a candidate tumor suppressor, was recently shown to act as a nuclear PI(5)P receptor a function that regulates its ability to activate p53 (Gozani et al., 2003). PI(5)P has also been tied to tumor suppression via its potential regulation of Akt. It was recently demonstrated that loss of PI(5)P, via conversion to PI(4,5)P2 by the phosphoinositide kinase PIP4K II, resulted in a decrease in Akt activity (Carricaburu et al., 2003). Lastly, PI(5)P has been shown to enhance the activity of various myotubularin phosphatases (MTM1, MTMR3, and MTMR6) toward their preferred substrate PI(3,5)P2, presumably through allosteric regulation (Schaletzky et al., 2003). Together these studies stress the importance of PI(5)P as a bona fide signaling molecule, and not merely a metabolic precursor to other PI's as once proposed.
Protein tyrosine phosphatase localized to the mitochondrion (PTPMT1) is a member of the PTP superfamily discussed above, characterized by their highly conserved CX5R (SEQ ID NO:2) catalytic motif. Mitochondria are ubiquitous and dynamic organelles that house many crucial cellular processes in eukaryotic organisms. In addition to being responsible for the production of over 90% of cellular ATP through the TCA cycle and oxidative phosphorylation, mitochondria are the site of fatty acid oxidation, ketone body production, heme biosynthesis, cardiolipin metabolism, the production of coenzyme Q, ROS production, key steps of gluconeogenesis and the urea cycle, and are central to the mechanisms of apoptosis (Newmeyer and Ferguson-Miller, 2003; Voet, 2004). More specialized mitochondrial functions, including the coupling of glucose metabolism to insulin secretion in the pancreatic  cell, have also evolved in various tissues (Maechler and Wollheim, 2001). Disruption of these and other mitochondrial functions results in more than 40 known diseases, including Parkinson's, Alzheimer's and diabetes, further underscoring the importance of properly functioning mitochondria to human health (UMDF, 2004; Pestronka, 2004; Schon, 2000; Wallace, 1999).
Despite the level of attention given to mitochondrial function during the past approximately 50 years, there remains only a vague understanding of the role of phosphorylation within this organelle. To date, the phosphorylation and dephosphorylation of the E1 subunits of the pyruvate and branched-chain α-ketoacid dehydrogenase complexes (PDC and BCKD) constitute the only well-characterized examples of regulation by reversible phosphorylation within mitochondria (Harris et al., 1997; Roche et al., 2001). However, a number of studies suggest that phosphorylation is more widely important in mitochondrial physiology. Multiple groups have reported that the addition of radiolabeled ATP to purified mitochondria leads to the formation of an array of phosphoproteins, the number of which increases with the addition of phosphatase inhibitors. The recent introduction of a novel phospho-specific dye, allowing for the observation of steady-state phosphorylation, has further contributed to the list of potential mitochondrial phosphoproteins (Schulenberg et al., 2003). Approximately 25 of these proteins have been identified, including respiratory chain subunits and multiple members of the TCA cycle (Bijur and Jope, 2003; Bykova et al., 2003; Chen et al., 2004; Hojlund et al., 2003; Lee et al., 2004; Schulenberg et al., 2003). The discovery of mitochondrial kinases and phosphatases capable of regulating these phosphoproteins is thus of critical importance in determining the functional consequences of these phosphorylation events.
As part of our discovery, it was realized that PTPMT1 is among the most highly conserved phosphatases known, possessing orthologs in all four phylogenetic kingdoms, including the eubacterium Pirellula sp. strain 1. Additionally, PTPMT1 is the first member of the PTP superfamily to reside exclusively within the mitochondrion. PTPMT1 is directed to the mitochondrion by a cryptic N-terminal signal sequence where it colocalizes with members of the respiratory chain on the matrix face of the inner membrane. Disruption of PTPMT1 expression in the pancreatic P cell line INS-1 832/13 causes an approximate 80% increase ATP production, and markedly enhances insulin secretion under both basal and glucose stimulated conditions. This same treatment alters the mitochondrial phosphoprotein profile, suggesting that these observed biological effects may be due to a phosphorylation event or events involving PTPMT1. Together, these data suggest a possible new mitochondrial control point in the regulation of insulin secretion from β cells, and define PTPMT1 as a possible new drug target for the treatment of type II diabetes. Moreover, the discovery of a bona fide mitochondrial PTP further suggests that crucial mitochondrial functions are likely to be regulated by reversible phosphorylation.